What are DNA sequence motifs?

Patrik Dhaeseleer
Sequence motifs are becoming increasingly important in the analysis of gene regulation. How do we define sequence motifs,
and why should we use sequence logos instead of consensus sequences to represent them? Do they have any relation with
binding affinity? How do we search for new instances of a motif in this sea of DNA?

Patrik Dhaeseleer is in the Microbial Systems

Division, Biosciences Directorate, Lawrence
Livermore National Laboratory, PO Box 808,
L-448, Livermore, California 94551, USA
e-mail: patrikd@llnl.gov

a HEM13

CCCATTGTTCTC
TTTCTGGTTCTC
TCAATTGTTTAG
CTCATTGTTGTC
TCCATTGTTCTC
CCTATTGTTCTC
TCCATTGTTCGT
CCAATTGTTTTG

HEM13
HEM13
ANB1
ANB1
ANB1
ANB1
ROX1

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002700000010
464100000505
000001800112
422087088261

Bob Crimi

A
C
G
T
Counts

YCHATTGTTCTC

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Sequence motifs are short, recurring patterns

in DNA that are presumed to have a biological function. Often they indicate sequencespecific binding sites for proteins such as
nucleases and transcription factors (TF).
Others are involved in important processes at
the RNA level, including ribosome binding,
mRNA processing (splicing, editing, polyadenylation) and transcription termination.
In the past, binding sites were typically
determined through DNase footprinting, and
gel-shift or reporter construct assays, whereas
binding affinities to artificial sequences were
explored using SELEX. Nowadays, computational methods are generating a flood
of putative regulatory sequence motifs by
searching for overrepresented (and/or conserved) DNA patterns upstream of functionally related genes (for example, genes with
similar expression patterns or similar functional annotation). For a while, it seemed
like we had more computationally predicted
sequence motifs without a known matching transcription factor, than transcription
factors without a known binding sequence,
although large-scale efforts to analyze the
genome-wide binding of transcription factors using ChIP-chip are rapidly rectifying
this situation.
The abundance of both computationally
and experimentally derived sequence motifs
and their growing usefulness in defining
genetic regulatory networks and deciphering
the regulatory program of individual genes
make them important tools for computational biology in the post-genomic era.

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2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

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Figure 1 ROX1 binding sites and sequence motif.

(a) Eight known genomic binding sites in three
S. cerevisiae genes. (b) Degenerate consensus
sequence. (c,d) Frequencies of nucleotides at
each position. (e) Sequence logo showing the
frequencies scaled relative to the information
content (measure of conservation) at each position.
(f) Energy normalized logo using relative entropy to
adjust for low GC content in S. cerevisiae.

NATURE BIOTECHNOLOGY VOLUME 24 NUMBER 4 APRIL 2006

Restriction enzymes and consensus

sequences
Type II restriction enzymes, discovered in the
late 1960s, need to bind to their DNA targets
in a highly sequence-specific manner, because
they are part of a primitive bacterial immune
system designed to chop up viral DNA from
infecting phages. Straying from their consensus binding site specificity would be the
equivalent of an autoimmune reaction that
could lead to irreversible damage to the bacterial genome. For example, EcoRI binds to the
6-mer GAATTC, and only to that sequence.
Note that this motif is a palindrome, reflecting the fact that the EcoRI protein binds to
the DNA as a homodimer. Other restriction
enzymes bind to a degenerate consensus
sequence. For example, HindII bind to the
sequences GTYRAC, where Y stands for C
or T (pYrimidine), and R stands for A or G
(puRine). (See http://www.chem.qmul.ac.uk/
iubmb/misc/naseq.html#tab1 for a listing of
the IUPAC symbols for degenerate consensus
sequences.)
We can calculate how often we would
expect these consensus sequences to occur,
based on their length and degeneracy. The
probability that a random 6-mer matches
the EcoRI binding site is (1/4)6, so the site
occurs about once every 46 (= 4,096) bp in a
random DNA sequence. The HindII binding
site, containing two positions where two out
of four bases can match, would occur once
per 44 22 (= 1,024) bp.
Consensus or caricature?
Other DNA binding proteins tend to be less
picky in sequence specificity. In 1975, Pribnow
discovered the TATAAT box, a well-conserved
sequence centered around 10 bp upstream of
the transcription initiation site of Escherichia
coli promoters. This motif, together with a

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 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

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TTGACA motif centered around 35, forms
the binding site for the 70 subunit of the
core RNA polymerase. However, despite the
high degree of conservation at each position
(ranging from 54% to 82% for each base), it is
actually extremely rare to find a promoter that
matches this consensus sequence exactly, with
most promoters matching only 79 out of the
12 bases. Rather than representing a typical
binding sequence, the consensus sequence in
this case is instead a highly unusual sequence.
It turns out that the activity of each promoter
is related to how well it matches the consensus sequence, so the activity level of each gene
can be fine-tuned by how much its 10 and
35 regions deviate from the consensus.
A better description of the binding
sequence in this case is through a Position
Frequency Matrix (PFM). Rather than only
keeping track of the most common base at
each position, we record how often each
base occurs in known sites. For example,
the Rox1 transcription factor is known to
bind at least eight sites in three genes in the
Saccharomyces cerevisiae genome. Figure 1
shows the multiple alignment of these eight
binding sites, with a consensus sequence of
YCHATTGTTCTC. (Conventionally, a single
base is shown if it occurs in more than half
the sites and at least twice as often as the second most frequent base. Otherwise, a doubledegenerate symbol is used if two bases occur
in more than 75% of the sites, or a tripledegenerate symbol when one base does not
occur at all.) The frequency matrix and its
graphic representation in Figure 1 clearly
show a core motif of ATTGTT, with much
lower conservation in the flanking bases.

to be applied. This explains why the central

positions of the motif in Figure 1e show an
information content of less than 2 bits, even
though they are perfectly conserved within
the eight known binding sites.
Note that the total information content of
a motif is directly related to its expected frequency of occurring within a random DNA
sequence. For example, the information content of the partially degenerate 6-mer HindII
binding site is 10 bits (2 bits per conserved
base, 1 bit per double-degenerate position),
and its expected frequency in random DNA
is 1 in 210 = 1,024.

Sequence logos
By scaling each stack of letters in Figure 1d
with some measure of the conservation at
each base, we get a much clearer view of the
binding sequence. In a sequence logo, developed by Schneider and Stephens1, each stack
is scaled with the information content of the
base frequencies at that position:

where pb is the background frequency of

base b in the genome. This is equivalent to
a log-likelihood ratio (G test) to measure
the degree of disagreement between the
observed and background base frequencies,
and thus can again be used to calculate the
significance of the motif itself (and the frequency of occurrence of such a sequence in
random DNA).
Figure 1f shows the Rox1 binding motif,
corrected for the GC-content of S. cerevisiae genomic DNA using equation (2). In
comparison with e, the central G base now
carries more information than the flanking A
and T bases, reflecting the fact that its occurrence is much more significant in the low-GC
genome. The total information content of the
motif is Iseq = 11.27 bits.

Ii = 2 + fb,i log2 fb,i

b

(1)

where fb,i indicates the frequency of base b at

position i. Positions that are perfectly conserved contain 2 bits of information, those
where two of the four bases occur 50% of the
time each contain 1 bit, and positions where
all four bases occur equally often contain no
information. Note that for a small sample, the
information content will tend to be overestimated, so a small-sample correction needs

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Correcting for background frequencies

Equation (1) assumes all four bases occur
equally often in the background genomic
DNA. For organisms such as E.coli (51% GC)
or human (41%) this is usually a reasonable
approximation. However, for genomes with
a more biased GC content such as S. cerevisiae (38%), Caenorhabditis elegans (36%)
and especially extremes such as Plasmodium
falciparum (19%) or Streptomyces coelicolor
(72%), a correction factor is needed. One
approachadvocated by Schneideris to
replace the 2 in equation (1) with the lower
entropy of random DNA of the specified GC
content. A more informative approach2 is to
generalize equation (1) to the relative entropy
(a.k.a. Kullback-Leibler distance) of the binding site with respect to the background frequencies:
f
Iseq(i) = fb,i log2 pb,i
b

(2)

Roll your own logos

Two free web interfaces exist for generating
a sequence logo from your favorite DNA

alignment: Steven Brenners WebLogo

(http://weblogo.berkeley.edu/), implementing Schneiders original Sequence Logos,
and the more recent enoLOGOS3 (http://
biodev.hgen.pitt.edu/enologos), using relative entropy. The former provides an option
to put error bars on the information content, which can be quite useful especially for
motifs based on a small number of sequences.
However, the latter offers a wider variety of
input formats, variable GC content, and the
option to examine nonindependent bases via
mutual information. The two sites also take
a different approach to small-sample correction. The logos in Figure 1 were generated
using enoLOGOS.
Transcription factor binding sites are
collected in a number of online databases,
including TRANSFAC4 (http://www.generegulation.com/pub/databases.html),
JASPAR 5 for multicellular eukaryotes
(http://jaspar.genereg.net), YEASTRACT6
(http://www.yeastract.com/) and SCPD7
(http://rulai.cshl.edu/SCPD) for S. cerevisiae, RegulonDB8 for E. coli (http://
regulondb.ccg.unam.mx) and PRODORIC9
for prokaryotes (http://www.prodoric.de/),
although some of these are still focused primarily on consensus sequences.
Binding energy and searching for
novel sites
As mentioned above, the affinity of a DNA
binding protein to a specific binding site is
typically correlated with how well the site
matches the consensus sequence. However,
not all positions in a binding site are equally
forgiving of mismatches, and not all mismatches at a given position have the same
effect.
If we assume that each position contributes to the binding energy independently (a
reasonable approximation in most cases), we
could laboriously measure the effect on binding energy of all possible single base changes.
The resulting Position Weight Matrix (PWM)
W(b,i) can then be used to calculate the specific-binding free energy (relative to random
background DNA) of a sequence S as:
where S(i) is the base occurring in position i
in sequence S.
Gs(S) = W(S(i),i)
i

(3)

Typically, we only have a list of known

binding sites, without any affinity information. If we assume that the genomic DNA is
random with base frequencies pb, it is possible to optimize the values in the PWM such

VOLUME 24 NUMBER 4 APRIL 2006 NATURE BIOTECHNOLOGY

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that the probability of binding to the known
binding sites (versus the more abundant
background DNA) is maximized. The optimal weight matrix is then given by:
f
W(b,i) = log2 pb,i

(4)

2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

The information content Iseq can then be

interpreted as an estimate of the average
specific binding energy to the entire set of
known binding sites, in competition with the
genomic DNA.
This PWM can be used to search for novel
sites with high predicted binding affinity within the rest of the genome, typically
using a score threshold based on the scores of
the known binding sites. Unfortunately, this
approach can result in large numbers of false
positives, sometimes returning hundreds or

thousands of putative binding sites. A promising alternative developed by Djordjevic et

al.10 is to simultaneously optimize the weight
matrix as well as the threshold such that all
the known sites are included, but as few other
sites as possible, typically resulting in many
fewer and more reliable novel sites.
Nevertheless, it is unavoidable that
sequence motifs with low information content (that is, short motifs, and/or with a lot
of degeneracy) will tend to yield large numbers of fairly low affinity hits, especially
in large eukaryotic genomes. Presumably
other factors such as chromatin structure
and cooperative binding play a role as well
in determining the in vivo specificity of the
associated transcription factors.
1. Schneider, T.D. & Stephens, R.M. Sequence Logos:
a new way to display consensus sequences. Nucleic
Acids Res. 18, 60976100 (1990).
2. Stormo, G.D. DNA binding sites: representation and
discovery. Bioinformatics 16, 1623 (2000).

NATURE BIOTECHNOLOGY VOLUME 24 NUMBER 4 APRIL 2006

3. Workman, C.T. et al. EnoLOGOS: a versatile web tool

for energy normalized sequence logos. Nucleic Acids
Res. 33 (Web Server Issue), W389W392 (2005).
4. Matys, V. et al. TRANSFAC and its module
TRANSCompel: transcriptional gene regulation in
eukaryotes. Nucleic Acids Res. 34 suppl. Database
issue, D108D110 (2006).
5. Vlieghe, D. et al. A new generation of JASPAR, the
open-access repository for transcription factor binding
site profiles. Nucleic Acids Res. 34 suppl. Database
issue, D95D97 (2006).
6. Teixeira, M.C. et al. The YEASTRACT database: a tool
for the analysis of transcription regulatory associations
in Saccharomyces cerevisiae. Nucleic Acids Res. 34
suppl. Database issue, D446-451 (2006).
7. Zhu, J. & Zhang, M.Q. SCPD: a promoter database of
the yeast Saccharomyces cerevisiae. Bioinformatics
15, 607611 (1999).
8. Salgado, H. et al. RegulonDB (version 5.0): Escherichia
coli K-12 transcriptional regulatory network, operon
organization, and growth conditions. Nucleic Acids
Res. 34 suppl. Database issue, D394D397 (2006).
9. Munch, R. et al. PRODORIC: prokaryotic database
of gene regulation. Nucleic Acids Res. 31, 266269
(2003).
10. Djordjevic, M., Sengupta, A.M. & Shraiman, B.I. A biophysical approach to transcription factor binding site
discovery. Genome Res. 13, 23812390 (2003).

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